Feed compositions

ABSTRACT

The disclosure relates to methods and compositions for controlling the formation of methane in rumen fermentation through the use of compounds containing halogen bonded to carbon which is in turn attached directly to a functional group. Preferred compounds are amides, alcohols, ketones, sulfones, sulfoxides, carboxylic acids, amines, sulfonamides and phosphonic acid derivatives. The disclosure also relates to novel compounds including N-(2-hydroxypropyl) derivatives of di- and trihaloamides and the N-(2-hydroxyethyl) derivatives of trihaloacetamides.

United States Patent Inventors [72] Arthur A. Patchett;

Dale R. l-Ioll, Cranford, NJ.; Clarence S. Rooney, Beaconslleld, Quebec,Canada [2]] Appl. No. 738,801 [22] Filed June 21, 1968 [45] PatentedSept. 21, 1971 [73] Assignee Merck & Co. Inc.

Rahway, NJ.

[54] FEED COMPOSITIONS 5 Claims, No Drawings [52] US. Cl 424/320,260/557, 260/558, 260/561, 424/324 [51] lnt.CI ....A6lk 27/00 [50] Fieldof Search 424/320, 324; 260/561 X; 99/2, 2 N

[56] References Cited UNITED STATES PATENTS 2,419,888 4/1947 Nolan etal. 424/320 3,206,509 9/l 965 Nyquist et al.. 260/561 X 3,314,849 4/l967Hamm Primary Examiner-Albert T. Meyers Assistant Examiner--Frederick E.Waddell Attorneys-John Frederick Gerkens, .l. Jerome Behan and l.

Louis Wolk ABSTRACT: The disclosure relates to methods and compositionsfor controlling the formation of methane in rumen fermcntation throughthe use of compounds containing halogen bonded to carbon which is inturn attached directly to a functional group. Preferred compounds areamides, alcohols, ketones, sulfones, sulfoxides, carboxylic acids,amines, sulfonamides and phosphonic acid derivatives. The disclosurealso relates to novel compounds including N-(2-hydroxypropyl)derivatives of diand trihaloamides and the N-(2- hydroxyethyl)derivatives of trihaloacetamides.

FEED COMPOSITIONS BACKGROUND OF THE INVENTION 1. Field of the InventionThis application relates to novel methods for improving the efficiencyof utilization of feed elements in ruminants and to novel feedcompositions. It also relates to novel compounds and the processes formaking and using the same.

2. Description of the Prior Art The ruminant family of mammals ischaracterized by a digestive process wherein ingested feed materials aresubjected to an initial fermentation process in the rumen to giveproducts, notably fatty acids, which can be utilized for the nourishmentof the animal. Certain organisms such as Methanobacterium ruminantium,which are normally present in the rumen, produce methane rather than theuseful fatty acids as their major fermentation product. The methane isnot further utilized by the animal, and the carbon values representedthereby are lost, a loss which can approach I percent of the carbonvalues of the original feed. It is, therefore, desirable to minimize theproduction of methane in the rumen and to maximize the production ofdesirable lower fatty acids such as propionic and butryic acids in orderto realize the greatest possible efficiency from the feed supplied. Suchincreased efficiency results in a larger weight gain in the animal for agiven amount of feed, and reduced feed costs.

Heretofore, there has been no known method for suppressing methaneformation in the rumen and improving feed efficiency which has foundcommercial acceptance.

SUMMARY OF INVENTION The present invention provides a class ofhalogenated compounds which are useful in the suppression of methaneformation in the rumen. The invention also provides a method fortreating ruminants to maximize feed efficiency. There is furtherprovided novel feed and premix compositions and the method for using thesame.

The compounds which have been found useful, in ac-.

cordance with this invention, in suppressing methane formation,increasing feed efficiency and providing an increase in desirable lowerfatty acids are compounds which contain at least one chlorine, bromineor iodine atom, preferably at least two such atoms, attached to a carbonatom which is, in turn, attached to a single functional group. Thisstructure has been found characteristic of a high order of methanesuppressant activity. For example, the useful compounds can be amides,alcohols, carboxylic acids and esters, ketones, amines, sulfones,sulfoxides, sulfonamides, and phosphoric acid derivatives, eachcontaining at least one, and preferably at least two, chlorine, bromineor iodine atoms alpha to the designated functional group. The usefulcompounds are those wherein the noted halogen atoms are activated by asingle functional group, but are not sources of positive halogen such asdibromomalonamide wherein the attached halogen is activated by twofunctional groups.

The compounds which are useful in the practice of this invcntion includethe following:

a. amides of the general formula A& V wherein X is hydrogen, loweralkyl, monoearbocyclic aryl, lower cycloalkyl, chlorine, bromine oriodine; Y is hydrogen, lower alkyl, chlorine, bromine or iodine; Z ischlorine, bromine or iodine; and each of R and R is hydrogen, loweralkyl, lower cycloalkyl, monocarbocyclic aryl, heteroaryl, carbamyl orlower alkylene joined together or with the group X by carbon, oxygen ornitrogen to form a cyclic structure;

b. alcohols of the formula EFL. V 7

wherein Z is as defined above and the Z groups are the same ordifferent, and each of R and R is hydrogen, lower alkyl ormonocarhocyclic aryl;

c. ketones of the formula wherein X and Z are as defined above and the Zgroups are the same or different, and R is lower alkyl, monocarbocyclicaryl d. sulfones of the formula I X(ll S O;R a 2 wherein X, Y and Z areas defined above and R is lower alkyl, monocarbocyclie aryl or e.sulfoxides of the formula Y Xl- S O-Ru wherein X, Y, Z and R are asdefined above;

f. carboxylic acids and esters thereof of the formula Z X( J O-R7wherein X and Z are as defined above, the Z groups are the same ordifferent, and R is hydrogen or lower alkyl;

g. amines of the formula Z Ra Z(IJ-(IJNHQ wherein Z, R; and R are asdefined above and the Z groups are the same or different;

h. sulfonamides of the formula wherein X, Y, Z, R, and R are as definedabove;

i. phosphonic acid derivatives of the formulae 0 Z- i (NHRQ and aryl,halo-lower alkyl, nitro, monocarbocyclic aroyl, lower alkanoyl, carboxy,carbo-monocarbocyclic aryloxy, lower alkoxy, monocarbocyclic aryloxy,lower alkylthio, carbamyl,

lower alkyl carbamyl, monocarbocyclic aryl carbamyl, sulfonamido, amino,cyano, etc. Accordingly, the useful lower alkyl groups may be halo-loweralkyl, monocarbocyclic aryl lower alkyl, hydroxy lower alkyl, heteroaryllower alkyl, lower alkoxy lower alkyl, carbo-lower alkoxy lower alkyl,monocarbocyclic aryloxy, lower alkyl, lower alkanoyl lower alkyl,carbamyl lower alkyl, lower alkyl carbamyl lower alkyl, etc. Similarlysubstituted eycloalkyl groups can be employed. Monocarbocyclic arylswhich may be employed include haloaryl, halo-lower alkyl aryl, loweralkaryl, nitroaryl, carboxyaryl, lower alkoxy aryl, hydroxyaryl, hydroxylower alkyl aryl, aminoaryl, lower alkanoyl aryl, carbo-lower alkoxyaryl, sulfonamidoaryl, etc. Heteroaryl groups which may be used includelower alkyl heteroaryl, halo-lower alkyl heteroaryl, loweralkoxyheteroaryl, carboxyheteroaryl, aminoheteroaryl, lower alkanoylheteroaryl, carbo-lower alkoxy heteroaryl, etc.

Where the functional group of the active compound contains a replaceablehydrogen atom, it may bear an inert substituent. For example, amides maybe substituted by an amino group to form a hydrazide, or by a carbamylgroup to form a urea. Alcohols may be substituted by a lower alkyl groupto form an ether, or by a lower alkanoyl or monocarbocyclic aroyl groupto form an ester. Amines may be used in the from of nontoxic salts, suchas amine hydroehlorides. Carboxylic acids may be used in the form ofsoluble salts such as alkali metal or ammonium salts.

It is preferred that the total number of carbon atoms contained in eachof the groups X, Y, R R R R R R R and R be less than about 10. Higherlipophilic carbon chains tend to decrease the solubility of the compoundin the rumen fluids to a point where they are no longer present ineffective amounts due to insolubility.

A lower alkyl group, as that term is used herein, is an alkyl groupcontaining less than 10 carbon atoms. Specific examples of useful loweralkyl groups include methyl, ethyl, npropyl, isopropyl, n-butyl,secbutyl, isobutyl, amyl, hexyl, heptyl, octyl, nonyl, trifluoromethyl,2-chloroethyl, 2, 2- dichloroethyl, l-bromoethyl, 2-chloropropyl,2-hydroxyethyl, Z-hydroxypropyl, 3-hydroxypropyl, benzyl, Z-phenylethyl,2- phenylpropyl, nitrobenzyl, methylbenzyl, and similarly substitutedmethyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, amyl, hexyl,heptyl, octyl and nonyl radicals. Suitable specific cycloalkyls includecyclopentyl, cyclohexyl, cycloheptyl and incrtly substituted derivativesthereof. Specific heteroaryls include pyrryl, pyridyl, triazolyl,thiazolyl, pyranyl, furyl, pyrrolidyl, pyrimidyl, imidazolyl, andbenzimidazolyl. Useful monocarbocyclic aryls include phenyl,nitrophenyl, trifluoromethylphenyl, tolyl, xylyl, ethylphenyl andsimilarly substituted phenyl radicals. It will be understood that theabove-described substituents X, R R R R R and R can constitute abridging group in order to form dimeric compounds illustrated by thefollowing:

wherein a is integer.

The preferred X substituents are hydrogen, chlorine, bromine,unsubstituted lower alkyl, chloro-lower alkyl and bromo-lower alkyl. Thepreferred Y and Z substituents are chlorine and bromine. In all cases,it is preferred that the X, R R R R R R R and R moieties be free ofionicsubstituents such as quaternary ammonium, amidinium, free sulfonic acidor phosphoric acid groups, etc., since the presence of such ionicsubstituents tends to decrease markedly the activity of the compounds insuppressing methane formation in the rumen.

In general, the preferred classes of compounds for use in the inventionare the following, in which the halgen is chlorine or bromine:

a. diand trihalogenated amides b. trihalogenated alcohols e. diandtrihalogenated ketones d. mono-, diand trihalogenated sulfones e. mono-,diand trihalogenated sulfoxides f. trihalogenated carboxylic acids g.trihalogenated amines h. diand trihalogenated sulfonamides; and

i. diand trihalogenated phosphonic acid derivatives.

With regard to compounds of category a., those having at least onehydrogen attached to the amide nitrogen generally show the highestactivity, although the disubstituted amides are also active.

Compounds employed in the practice of this invention can be prepared byknown methods of synthesis. For example, ahaloamides are readilyprepared by the reaction of the cor responding amine and acyl halideaccording to the equation: (1) Z 0 R1 Z (1? R1 R1 x-bi J-o1 zNfi x-tx-o-N NILIICL or by the reaction of a cleavable ester such as a methylor phenyl ester with an amine according to the equation:

The sulfonamides and phosphonamides can be prepared by substituting theappropriate sulfonyl or phosphonyl chloride for the acyl chloride inreaction l The trihalogenated amines and alcohols are all readilyavailable compounds or simple derivatives thereof. The carboxylic acidsare similarly available or readily prepared and the corresponding estersare made by direct esteriflcation or by converting the acid to the acylhalide or anhydride, followed by reaction with the desired 5 alcohol.

Sulfoxides and sulfones are prepared by progressive oxidation of thecorresponding sulfides, suitably by hydrogen peroxide, according to theequation:

where the ultimate sulfoxide or sulfone is an alphamonohalogenatedcompound, the sulfide starting material can be prepared byhaloalkylation of the corresponding mercaptan, according to theequation:

cohol in the presence of an acid acceptor such as a tertiary amine,according to the equation:

The invention also provides certain novel halogenated N- hydroxyalkylamide compounds which are especially suitable for use in improving theefficiency of feed utilization in ruminants. These novel compounds arecharacterized by the structures:

and

Similarly, the N-(2-hydroxyethyl) alpha trihaloamides are prepared as inequations (6), (7), and (8) by using a trihalo acyl halide or ester and2-aminoethanol. In place of any of the acyl halides, there may be usedthe free acid or an anhydride thereof. In equation (8), other cleavableesters such as the ethyl, propyl or phenyl ester can be employed.

Preparation of the novel compounds of the invention may be illustratedby the following illustrative examples: Example l-N:(2-hydroxypropyl)trichloroacetamide Trichloroacetyl chloride (18.2 gm.) is dispersed in50 ml. of chloroform and the solution is cooled in an ice bath. Asolution of 2-aminopropanol ([5 gm.) in 20 ml. of chloroform is addedwith stirring over a period .of about lO'minutes. An additional flt) ml.of chloroform is added and the mixture is stirred for about 2.5 hours.The solution is then separated from the oily hydrochloride, washed withwater and dried over sodium sulfate. The solution is evaporated'todryness, and the solid residue is recrystallized from benzene to give6.2 gm. of N-(2-hydroxypropyl) trichloroacetamide, melting point 66-69C. and containing 27.63 percent C (27.23 percent theory); 3.66 percent H(3.67 percent theory); 6.20 percent N (6.35 percent theory); and 47.56percent Cl (48.24 percent theory).

By substituting dichloroacetyl chloride for trichloroacetyl chloride,there is prepared N-( Z-hydroxypropyl) dichloroacetamide, melting point69-70 C. Example 2-N-(2- hydroxyethyl) trichloroacetamideTrichloroacetyl chloride (18.2 gm.) is dissolved in 50 ml. ofchloroform. The solution is cooled in an ice bath and a solution of 12.2gm. of 2-aminoethanol in 20 ml. of chloroform is added, with stirring,over about 10 minutes. An additional 30 ml. of chloroform is added andthe mixture is stirred for about 2.5 hours. The crystallinehydrochloride is filtered off and the filtrate is taken to dryness. Theresidue is recrystallized from benzene to give 4.25 gm. ofN-(2-hydroxyethyl) trichloroacetamide, melting point 55-57.5 C. andcontaining 23.61 percent C (23.56 percent theory); 2.88 percent H (2.93percent theory); 6.85 percent N-(6.78 percent theory); and 50.l7 percentCI (51.51 percent theory).

These novel compounds possess high activity together with high stabilityand low volatility. It is thus possible to prepare feed compositions andpremixes containing the compounds which are unaffected by long periodsof storage and do not lose activity through evaporation. They are alsosoluble in rumen fluids in effective amounts. The 2-hydroxypropyl amidederivatives are appreciably more resistant to enzymatic cleavage in therumen than the isomeric 3-hydroxypropyl derivatives (as shown bycarbon-l4 tracer studies). In addition these novel amide derivativesalthough absorbed through the rumen wall, are absorbed at a slower ratethan that of amide derivatives possessing less polar substituents. Thusthe combination of greater resistance to enzymatic cleavage andrelatively slow absorption through the rumen wall results in thepersistence of these amide derivatives in rumen fluids in an activestate for a significant period of time. When metabolized by theruminant, the novel compounds yield halo acids which are readilyexcreted in the urine, and an aminoalcohol which is known to occurnaturally.

Activity of the described compounds in inhibiting methane formation,increasing the proportion of desirable propionic and butyric acids andgenerally improving the efficiency of feed utilization in ruminants canbe demonstrated by both in vitro and in vivo tests. Testing of allcompounds by long term feeding tests in representative animals isprohibitive both in time and in the amounts of feed and additivecompounds consumed. It has been found that fermentation processesequivalent to those which occur in the rumen can be established underlaboratory conditions and that the results obtained from such laboratoryfermentations correlate closely with actual experience in ruminants withrespect to feed utilization, and particularly with respect to methanesuppression, production of fatty acids and toxicity to the rumenorganisms.

The artificial rumen tests which are known to correlate with animalstudies and are those employed herein are of three types. In all tests,the principal component of the fermentation medium is rumen fluidcollected from fasted sheep and strained through multiple layers ofsterile cheesecloth. The medium is buffered to pH 6.8 and maintained at39 C.

throughout the test period. The test vessel is flushed with inertmilliliter. Gas samples are collected over a period of 2.5 hours and thegas is analyzed for methane. The following compounds are found to giveessentially 100 percent suppression of the formation of methane in thefermentation over the test period:

1. dichloroacetamide 2,2-dichloropropionamide 2-bromopropionamide2,2,3-trichloropropionamide N-p-chlorophenyl chloroacetamide2-bromo-2-methylpropionamide .chloroacetamide dibromoacetamide2-chloropropionamide 10. trichloroacetamide ll.N-cyclohexyl-2,2-dichloropropionamide l2. N2-(p-nitr0phenyl)ethyldichloracetamide l3. N,N-dimethyl dichloracetamide 14. bis(dichloroacetamide) ethane l5. N-dichloroacetyl serine anilide l6.N-(2-carboxy-5-aminophenyl)dichloroacetamide l7.N-(m-ethoxy-p-carboxyphenyl)dichloroacetarnide l8.N-(3-chloro-5sulfonamidophenyl)dichloroacetamide [9. N-(2-thiazolyl)-2,2-dichloropropionamide 20.N-(p-tolyl)-2,2-dich1oropropionamide 2 l. N-(p-nitrophenyl)-2,2-dichloropropionamide 22. N-(4-pyridyl)-2,2-diehloropropionamide23. N-(p-carboethoxyphenyl)-2,2-dich1oropropionamide 24.N-benzyl-Z,2-dichloropropionamide 25.N-(Z-pyrimidyl)-2,2-dichloropropionamide 26 N-[ 1-(p-[methylthio]benzoyl)-2-hydroxyethyl]dichloroacetamide 27. N- [p-(methylthio)benzoylmethyl ]dichloroaeetamide 28. N-(p-carboxyphenyl)-dichloroacetamide 29. N-( N ,N '-dimethylaminoethyl)-2,2-dichloropropionamide hydrochloride 30. N-methyl chloroacetamide3|. N-henzyl ehlOtQiEWIHlidQ 32. p-nitro-diehloroacetanilide2,2-dichlorobutyramide N-dimethylamino-Z,2-dichloropropionamide2,3-dichloropropionamide DiiodoacetamideN,N-dimethy1-2,2-dichloropropionamide 4-(2,2-dichloropropionyl)morpholine N-(2-thiazoly1)diehloroacetamide 2,2-dichlopropionyl urea 4l.p-2,2-dichloropropionobromoanilide 42. 2,2-dichloropropionanilide 43.2,2-dichloro-2-phenylacetamide 44. Tribromoacetamide 45.2,2-dibromoprpionamide 46. N-cyclohexyl dichloroacetamide 47.N-eyclohexyl trichloroacetamide 48. 1,2-bis (2,2-diehloropropionamide)ethane 49. N-methyl-2,2-dichloropropionamide 50. 2',6-dimethyl 2,2-dichloropropionanilide 5|. N-cyclohexyl dibromoacetamide 52.2,2-dichloropropionyl thiourea 53. m-2,Z-dichloropropionaniside 54.1,2-bis (dibromoacetamido) ethane 55. N-[p-(l-methyl-5-nitro-2-imidazolyl) ylldichloroacetamide 56.N-cyclohexyl-2,2-dibromopropionamide 57. 1,2-bis(2,2-dibromopropionamide) ethane 58. 2,2-dibromopropionyl urea 59.2,2-dichlorobutyryl urea 60. 2,2-dibromoacetyl urea 61.2,2-diehloroacety1 urea 62. N-cyclohexyl tribromoacetamide 63.N-ethyl-2,2-dibromo-2-cyclohexyl acetamide 64.N-cyclohexyl-2,2-diehlorobutyramide 65. 1,3-bis(2,2-dichloro-propionamido)propane V 66. N(t-butyl)-2,2-dich1oropropionamide phen- 67.N-acetyl-2,2-dichloroacetamide 68. 1,6-bis (2,2-dichloropropionamido)hexane 69. p-hydroxy 2,2-dichloropropionanilide 70.N-benzyl-2,2-dichloroacetamide 71. N,N-bis (2,2-dichloropropionyl) urea72. 1,5-di (2,2-dichloropropionyl) biuret 73. N-[p(carbomethoxymethylcarbamyl) phenyl]-2,2-

dichloropropionamide 74. 1,8-bis (dichloroacetamido) n-octane 75.N-(n-butyl)-2,2-dichloropropionamide 76. 1-(2,2-dichloropropionyl)biuret 77. p-carboethoxyphenyl dichloroacetamide 78.N-(2,2-dichloropropionoxycthyl)-2,2-

dichloropropionamide H-1,32-oxazaphosphorine-Z-oxide 103. N-(2-hydroxypropyl) trichloroacetamide 104. N(2-hydroxyethyl)trichloroacetamide 105. N-hydroxymethyl-Z,Z-dichloropropionamide 106.2,2,3-trichloropropionyl urea l07. N,N-bis (dichloroacetyl) urea 108.N-(2,3-dihydr0xypropyl) dichloroacetamide 109. N,N'-bis(dichloroacetyl)-2-hydroxy-1,3 propylenediamine l 10.N-(1,3-dichloro-t-butyl)dichloroacetamide l 1 1.N-(diehloroacetamidomethyl) pyridinium chloride 1 12. N-(2-carbamoylexyethyl) dichloroacetamide l 13.N-(dichloroacetamidopropoxyethoxyethoxypropyl) dichloroacetamide 114.N-[3-(di-hydroxyethylamino) propyl]dichloroacetamide 115.3-carbamy|-l-(dichloroacetamidomethyl) pyridinium chloride 1 16.chloride 1 17. N-methylsulfonylmethyl dichloroacetamide 1 l8.N-2-(p-nitrobenzoyloxyethyl)dichloroacetamide 119.3-carboxy-1-(dichloroacetamidomethyl) pyridinium chloridenicotinic acidcomplex 120. Dichloroacetamidoethyl tetramethylphosphorodiamidate 121.N-2-chloroethyl dichloroacetamide 122. poly Dichloroacetyl derivative oftetraethylene pen taminc 123. N-(Hydroxyethoxyethoxypropyl)dichloroacetamide 124. N-chloromethyl dichloroacetamide 125.benzimidazole 126. N-6-hydroxy- 1 -hexyl) dichloroacetamide 127.2-(dichloroacetyloxyethyl) ammonium tosylate 128. N-[2-(N-morpholino)ethyl] dichloroacetamide 3-cyano-1-(dichloroacetamidomethyl) pyridinium2-(4-thiazolyl )-4(5 )-dichloroacetamido,

129. 2-(dichloroacetamidoethyl) nicotinate 130. bis (dichloroacetamido)methyl ether 13 l. tris (dichloroacetyl) melamine l 32.2,2,2-trichloroethanol 133. l-ethoxy-2,2,2-trichloroethanol 134.2,2,2-trichloro-t-butanol 135. hcxachloroacetone 136. di (chloromethyl)sulfone l37. di (chloromethyl) sulfoxide I38. methyl dichloromethylsulfone 139. N-phenyl dichloromethanesulfonamide 140.dichloromethanesulfonamide l4l. 2,2,2-trichlorethyl-3,5-dinitrobenzoatel42. 3,3-dichloro--phenyl-2-piperidonc I43. 1,],l-trichloro-3-methoxy-2-propanol 144. l l l-trichloro-2,3-propylcneglycol I45. l,l,l-trichloro-3-ethoxyisopropanol146. dicthyl trichloromethanephosphonatc 147. trichloromethanephosphonicacid dianilide I48. poly (3,3,3-trichloropropylene oxide) (M.W. about 9149. 2-tt'ichloromethyl-tetrahydro-ZH-l ,3,2-oxazaphosphorine-Z-oxide150. N,N '-bis-(dichloromethanesulfonyl)- l ,8-octanediamine l5l.1-(3-ethoxy-l,l,l trichloroisopropanol The foregoing compounds arerepresentative of the broad class of compounds hereinbefore defined andsubstantially the same results are obtained when any of the various X,Y, Z, R,, R R R R R R and R groups, as previously described, issubstituted for a corresponding group of the enumerated compounds.

In the second series of tests, the test compound is added to thestandardized artificial rumen fermentation at several dosage levels andthe inoculated fermentation medium is incu bated at 39 C. for 24 hoursagainst a control fermentation containing no test compound. Periodicagitation is provided throughout this period. At the conclusion of thetest, gas samples are collected and analyzed, the percent reduction inmethane produced, compared to the control, is plotted againstconcentration of test compound, and the dosage level required to producea 50 percent reduction in methane production is calculated. These valuesare useful in comparing the relative activities of the test compound.Data obtained in this manner are presented in the following table,wherein the test compounds are identified by number in accordance withthe enumeration hereinbefore given:

trichloroisopropoxy)-3,3,3-

Cumpound No. Amount Required For SOReduction (ug. per ml.)

1 3 2 43 3 l5 4 8 5 3l 6 l2 7 3 8 l 9 55 IO 8 l l 5 l2 l7 l3 I25 l4 5 l55 16 26 I7 16 l8 5 l9 i1 20 l l 21 80 22 13 2'3 l2 24 5 25 r. i 2o 10 NNu tac noutvruaumvt Amount Required For 50 Reduction pg. per ml.)

Compound No.

Following the procedure described above, various test compounds areadded to standardized rumen fermentations and are incubated for 24 hoursagainst a control. At the end of 24 hours, samples of the fermentationfluid are collected and analyzed for volatile fatty acid content.Results of these analyses are presented in the table below as the ratioof acetic acid to the total of propionic, butyric and valeric acids inthe rumen fluids. Thus, a reduction in this ratio indicates that thetest compound is effective in shifting the volatile fatty acidcomposition to the more desirable propionic, butyric and valeric acids.Measurements are made of the total amount of methane and total fattyacids produced in both the control and experimentalfermentations.

Concentration CH Ratio Compound g/ml.) Produced7.,2-dichloropropionamide 12 0.79 7 0.41 70 0 0.3 l diehloroacetamide0.73 7 0 0.40 13 0 0.48 2,2-dichloro-N -cyclohexy1 8 0.94

propionamide l l 2 0.52 22 0 0.42 2,2-dichloro-N -(p41'iflu0r0- 8 0.94

mcthylphenyl) propionamide 14 4 0.60 29 2 0.53 2,2-dichloro-N benzyl 101.01

propionamide 12 3 0.57 3 3 0 0.55 2,2,3-trichloropropionarnide 10 1.01 98 0.91 1 8 6 0.82 bis (dichloroacetamido) ethane 12 1.02 14 2 0.52 28 00.49 2,2-dichloro-N -(2-thiaz0ly1) 12 1.02 propionamidc ll 8 0.76 23 40.59 2,2 dichloro-N-(4-pyridyl) 9 1.07 propionamidc 11 7 0.61 22 5 0.482,2-dichloro-N -(p-carbethoxy- 9 1.07

phenyl) propionamide 1S 8 0.53 29 S 0.58 2,2,2-trichlorocthanol 9 0.73 89 0.69 0 0.39 dibromoacctnmidc 0 0.73

Table -Lkyntinued trichloroacetamidc N-cyclohexyl-Z,Z-dichloroacetarnide 2,2-dichloropropionyl urea diindoacctamidc2,2-dicltlorohutyramidc N,N-dimcthyl-2,2-dichloropropionamidetribromoacctamide 2,2-dibromopropionamide 2.2-dichloro-3-phenylacetamide 2,2-dichloropropionanilide2,6'-dimethy12,2-dichloropropionanilide N-cyclohexyl trichloro- 1,2-bis(2,2-dibromopropionamido) ethane N-cyclohexyl-Z,Z-dibromoacetamidedi(chloromethyl) sulfonc methyl dichloromethyl sulfone N,N'-bis(dichlomacetyl) LB-octane diamine N-p-carboethoxyphenyl-Z,2-

dichloroacetamide m-2,2dichloropropionophenolide 2,2-dichlorobutyrylurea N,N'-bis (2.2-dichloropropionyl) urea 2,2-dibromopropionyl ureaN-(methylsulfonylmethyl) N-( Z-cai'bamoyloxyethyl) dichloroacetarnideN-(p-nitrobenzoyloxyethyl) dichlomacetamidc 3 ,3 ,3-trichlcropropylcneglycol 1,1,l-trichloro-3-methoxy-2- propanol in order to provide a stilllonger term study of the effects of the test compounds on rumenfermentation, continuous cultures of the type described by Slyter et al.in Applied Microbiology, Vol. 12, pages 374-377 (1964) and Rufener etal. in Applied Microbiology, Vol. I 1, pages 196-201 (1963) areestablished and test compounds are added thereto. Comparison of thetreated fermentations with the controls provides a measure of theeffectiveness of the test compounds in eliminating methane anddecreasing the ratio of acetic acid to the total of propionic, butyricand valeric acids (calculated as above) in long term continuous rumenfermentations. The results of this series of tests are shown in thefollowing table.

All of the test compounds gave essentially complete inhibition ofmethane production at the levels used.

acetamide The foregoing data demonstrates the effectiveness of thedisclosed classes of compounds in reducing methane formation anddecreasing the ratio of acetic acid to propionic, butyric and valericacids in rumen fermentation. in preferred embodiments, essentiallycomplete inhibition of methane, together with a significant improvementin the proportion of higher volatile fatty acids is achieved. Anincrease in total energy content of fatty acid products is alsoachieved.

Correlation of the above-described rumen fermentation tests with animalstudies is demonstrated by data obtained from animal feeding tests suchas those described in the following examples:

Example 3-A 6-week animal feeding study is carried out by maintainingthree separate groups of sheep on diets of untreated alfalfa pellets,alfalfa pellets containing 0.006 percent by weight ofN-(Z-hydroxypropyl) dichloroacetamide, and alfalfa pellets containing0.024 percent by weight N-( 2-hydroxypropyl) dichloroacetamide,respectively. The amount of feed consumed by each test animal iscontrolled and recorded and each animal is weighed weekly.

At the conclusion of the test period, the amounts of feed consumed andthe total weight gain for each animal is computed and the averages foreach group are calculated. It is found that the control animals feeduntreated feed gain an average of 1 1.4 pounds per 100 pounds of feedconsumed. By comparison, animals fed a diet containing 0.006 percent ofthe test compound gain an average of 12.4 pounds per 100 pounds of feedconsumed and those fed a diet containing 0.024 percent of the testcompound gain an average of 12.8 pounds on the same amount of feed.Thus, it is apparent that the use of N-(Z-hydroxypropyl)dichloroacetamide provides a significant improvement in feed efficiencyin the test animals.

Example 4-Five wethers and one ewe ranging in age from 19 to 26 weeksand in weight from 40 to 60 Kgm. are each drenched with 2 gm. ofthiabendazole. Each animal is handfed to appetite twice daily, with acontrol ration of alfalfa pellets for a period of 8 weeks.

At the completion of the initial 8-week period, a portion of the testgroup is switched to a pelleted feed composition prepared by blendingnine parts of the same pelleted alfalfa used in the control feeding withone part of alfalfa premix containing 3.4 gm. of2,2-dichloropropionamide per kilogram of premix. The final feed thuscontains 0.034% by weight of the test compound. The remaining animalsare maintained on the untreated pelleted alfalfa. Each control andexperimental animal is hand-fed a limited ration of feed, twice daily.Samples of rumen fluids are taken and analyzed daily and weights arerecorded weekly.

At the end of 8 weeks, it is found that the sheep maintained on alfalfapellets containing 2,2-dichloropropionamide have an average daily weightgain of 139 gm. against an average daily gain of l 12 gm. for thecontrol animals. The test animals gain an average of 8.3 pounds per bodyweight per pounds of feed consumed while the control animals gain onlyan average of 6.3 pounds per 100 pounds of feed. For the treatedanimals, the average volatile fatty acid ratio in the rumen is 62:28:9(aceticzpropioniczbutyric), whereas the average ratio for controlanimals is 68:23:8. The ratio of propionic acid to acetic acid is 0.46for the treated animals and 0.34 for the controls. Thus, animals fed thetreated feed gain more weight than those fed the untreated feed, andshow an improvement in feed efficiency as well as a shift in volatilefatty acid composition to the more desirable propionic acid. g

In accordance with this invention, the compounds described areadministered orally to ruminants, either separately or admixed withfeed. Incorporation of the compound in the animals feed is the preferredmode of administration because of convenience and because it ensuresthat the compound and the feed material will be present simultaneouslyin the rumen. Precise dosages will vary depending on a number offactors, including the relative activity of the compound employed, thefeed material used, age of the animals, etc. In general, concentrationsof active compound of the order of about 0.005 percent to about 0.05percent of the total feed composition are found satisfactory. By basingthe dosage on concentration in the feed, the daily dosage isautomatically regulated with regard to body weight and age by theanimal's appetite. The feed compositions will, of course, also containat least one element of the ruminants diet, the element or elementspresent being those known to be suitable for the particular ruminant.Typical useful feed elements include grains such as barley, mile, andcorn; cellulosic roughage components such as hay, alfalfa, straw orcereal brans; vegetable and animal oils such as fish oil or soybean oil;nutritional supplements such as vitamin, mineral and nitrogenouspreparations, cottonseed meal, soybean meal; and stabilizers such aspreservatives or antioxidants. The precise components chosen will dependalso on local conditions such as availability of particular feedstuffs.at reasonable prices, pasturage and the like. A specific example of asuitable cattle feed could include:

50% shelled corn 35% alfalfa meal 10% soybean meal (44% protein) 5%molasses 0.24% N-(Z-hydroxypropyl) dichloroacetamide it is generallymost convenient to employ the compounds in the form of a concentratedpremix containing at least one element of the ruminants diet and anamount of the active compound of the order of about 0.05% to about 1.0%by weight of the total premix. The premix can also include such dietadditives or supplements as vitamin supplements, growth promoters andthe like. Final feed compositions are prepared by blending togethersuitable amounts of premix and other feed components, say one part ofthe former to nine parts of the latter. 1

In the foregoing description, the invention has been described byreference to certain specific illustrative embodiments thereof. It willbe understood, however, that the invention is not limited to thespecific embodiments described but encompasses all such modificationsand variations as fall within itsgeneral scope.

NAN. Ana-I What is claimed is:

l. A method for suppressing methane formation in the rumen of ruminantswhich comprises administering orally to said ruminant a methanesuppressing amount of at least one N-hydroxyalkylamide compound selectedfrom the group consisting of 1 and wherein X is hydrogen, loweralkylhaving from 1-9 carbon atoms, lower cycloalkyl having from 5-7 carbonatoms, phenyl, nitrophenyl, methylphenyl, ethylphenyl,trifluoromethylphenyl, chlorine, bromine or iodine and Z is chlorine,bromine, or iodine. v

. 2. A ruminant feed composition comprising at least one element ofruminant nutrition and a methane-suppressing amount of at least onecompound selected from the group consisting of Z O X( 3( 3-NHCH CH(OH)CH and 0 ll .7 A i fFBTQE H and wherein X is hydrogen, loweralkyl havingfrom 1-9 carbon atoms, lower cycloalkyl having from 5-7 carbon atoms,phenyl, nitrophenyl, methylphenyl, ethylphenyl, trifluoromethylphenyl,chlorine, bromine, or iodine and Z is chlorine, bromine, or iodine.

4. The composition of claim 3 wherein said N-hydroxy alkyl amidecompound is N-( Z-hydroxypropyl) dichloroacetamide.

5. The composition of claim 3 wherein said N-hydroxyalkylamide compoundis N-(2-hydroxypropyl) trichloroacetamide.

2. A ruminant feed composition comprising at least one element ofruminant nutrition and a methane-suppressing amount of at least onecompound selected from the group consisting of
 3. A ruminant feedsupplement premix composition comprising at least one element ofruminant nutrition having dispersed therein from about 0.05% to about1.0% by weight of at least one N-hydroxyalkyl amide compound selectedfrom the group consisting of
 4. The composition of claim 3 wherein saidN-hydroxy alkyl amide compound is N-(2-hydroxypropyl) dichloroacetamide.5. The composition of claim 3 wherein said N-hydroxyalkylamide compoundis N-(2-hydroxypropyl) trichloroacetamide.